Multiple primer extension

Modification experiments and primer extension analyses were performed on RNA from all four plant species. Representative gels are shown in Fig. 4. A large number of stops occur in the native RNA without DMS treatment. This phenomenon has been observed reproducibly in multiple RNA preparations using a variety of polymerization temperatures, and it may be due to a strong secondary structure within the rRNA that cannot be sequenced by reverse transcriptase. Also, there are many naturally occurring modified base residues within the rRNA sequences that can impede reverse transcriptase.23 Another possibility is that a portion of the RNA in each preparation is degraded at specific sensitive sites, and thus chain terminations result. Similar patterns of termination have been ob-... 

With regard to regulation of the human gene, primer extension studies indicated predominant transcription initiation sites in liver, lung, and kidney, and placenta 250 and 249 bp upstream from the translation start site and a second major site at — 100 bp, with the absence of TATA and CAAT patterns and a GC-rich sequence in the promoter region. Multiple (rat) testis-specific transcripts arise from differential polyadenylation site usage . 

Ross and coworkers were the first to describe the determination of ratios of primer extension products by MALDI-TOF-MS for the analysis of DNA mixtures [211]. This approach opened the door to new means of applying MALDI-TOF-MS to DNA analysis, notably in cases that included quantitative abilities. The main application for this approach-the relative quantitation of allele frequendes-is achieved by calculating the ratio of the peak area associated with aUele-spedfic extension products. The combined allele frequency of a biallelic SNP in a sample pool (and in an equimolar mixture of genomic DNA from multiple individuals) is... 

Figure 3.25 Polymerase chain reaction. The steps involved in the chain reaction are as follows (i) Incubation of the DNA at a temperature above 90 °C in order to separate the two strands of the DNA duplex, (ii) Cooling of the solution to about 50 °C to allow annealing of the primers to the template (i.e. the nucleotides bind to the template DNA according to the basepairing rules), (iii) Finally, addition of the polymerase and Mg ions to extend the nucleotide primer and complete the synthesis of the complementary DNA, which takes place at about 70 °C. (iv) The sequence (i) to (iii) is repeated to allow another extension to occur many repetitions can be carried out which results in enormous multiplication of the DNA strands. NTPs - deoxyri-bonucleoside triphosphates.

Fig. 4.1. Schematic representation of die StEP process using two parental DNA sequences. (1) Denatured template DNAs are primed widi defined primers. (2) The partially extended primers produced by very brief annealing/extension randomly reanneal to different parent sequences (template switching). (3) Novel recombinants are created through multiple cycles of annealing/extension and strand switelling, hi principle, StEP is also an error-prone amplification process dial introduces additional point mutations (white circles).

An extension to this existing kit is the QuikChange Multi, which enables multiple mutations at different sites. It uses only one mutated oligonucleotide primer, in contrast to the kit described above, but follows the same procedure using the restriction enzyme Dpnl. The single-stranded plasmid is converted into a double strand in vivo after transformation into an E. coli host. 

Synthesis of a specific DNA segment via the PCR is directed by two oligonucleotide primers. Multiple rounds of extension from the primers amplify the target sequence exponentially, with termini defined by the 5 ends of the two primers. This process allows the determination of sequences from a very small number of target molecules. Unique sequences can be amplified from a complex mixture of nucleic acids because of the high specificity of the primer-template hybridization. 

The assay described herein is capable of detecting at least three of the multiple functions of the polymerase. These are the initiation of DNA synthesis, the translocation of the initiation DNA-po/ complex, and the extension of the genomic negative-strand DNA using a portion of the pregenomic RNA as template. The initiation of DNA synthesis by DHBV pol is unusual in that the polymerase protein acts as a primer and the first nucleotide incorporated (dGTP) is covalently linked to a tyrosine residue near the N-terminus of the... 

Figure 5 Effect of BSU-1051 on the time-course of telomerase activity using (A) the 18-mer telomeric primer d[TTAGGG]3 (1 /iM) without (left-hand lanes) or with (right-hand lanes) BSU-1051 added at 10 /uM (76). The boxes identify the 40-min samples, which show altered multiple band patterns due to 3 -exonuclease activity. (B) Time-course of total amount of [o -32P]-dGTP incorporated into the extension products of the d[TTAGGG]3 primer in the presence and absence of BSU-1051. (C) Time-course incorporation of [a-32P]-dGTP into the 22-mer, 28-mer, and 34-mer and comparison of patterns of sets of multimers for the 22-mer and 28-mer in the presence and absence of BSU-1051. The diagrams between the two sets of results show the proposed structures of the species formed at each step.

Fig. 13. Schematic of the single-base extension assay applied to Tag probe arrays. Regions containing known SNP sites (A or G in this example) are first amplified by PCR. The PCR product serves as the template for an extension reaction from a chimeric primer consisting of a 5 tag sequence and a 3 sequence that abuts the polymorphic site. The two dideoxy-NTPs that could be incorporated are labeled with different flurophors in this example, ddUTP is incorporated in the case of the A allele, and ddCTP for the G allele. Multiple SBE reactions can be done in a single tube. The tag sequence, unique for each SNP, directs the extension products to a particular address on the Tag probe array. The proportion of a fluorophor at an address reflects the abundance of the corresponding allele in the original DNA. (Reprinted with permission from [45])...

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